Caldesmon, an actin- and calmodulin-binding protein found in smooth muscle and many non-muscle cells, is thought to be involved in the regulatory process of smooth muscle contraction, as well as other types of cytoskeletal movements. The fact that caldesmon is phosphorylated upon stimulation, that it is present in subplasmallemal regions of the cells, and the recent finding that it also interacts with phospholipids raise the possibility that caldesmon could play an even more intriguing role in the signal transduction process. The experiments described in this application is aimed to examine this possibility. The overall objective of the proposed research is to determine whether caldesmon is involved in transmembrane signalling pathways in smooth muscle and non-muscle cells. There are three specific aims: (1) to determine the binding site(s) of phosphatidylserine on caldesmon; (2) to establish whether caldesmon binds to isolated smooth muscle membranes, either to the lipids or to the protein constituents; and (3) to determine the factors' regulating transient association of caldesmon with membranes. The significance of the ability of caldesmon to interact with phospholipids and its relationship with the fact that caldesmon is able to assemble G-actin into filaments will also be investigated. A number of physico-chemical and molecular biology techniques will be used, including fluorescence spectroscopy, chemical modification of proteins, affinity column chromatography, site- directed mutagenesis, and the preparation of synthetic peptides. The results of these studies will not only provide information on the physiological roles of caldesmon, but may also lead to a better understanding of the regulatory process of smooth muscle contraction and cellular signal transduction. This project is a logical expansion of an ongoing research program entitled "Molecular Mechanism of Smooth Muscle Regulation". Dr. Dabrowska's team was chosen to be the collaboration partner because of her demonstrated expertise in the field of caldesmon, and her more recent work on the interaction between caldesmon and phospholipids. The collaborative arrangement combines the complementary expertise of both laboratories. This project will not only enhance the research program of the applicant, but will also benefit the scientific interest of Dr. Dabrowska.